Cartilage mosaic compositions and methods

ABSTRACT

Compositions comprising a cartilage sheet comprising a plurality of interconnected cartilage tiles and a biocompatible carrier are provided. Methods of manufacturing cartilage compositions comprising a cartilage sheet comprising a plurality of interconnected cartilage tiles are also provided.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims benefit of priority of U.S. ProvisionalApplication No. 61/768,190, filed Feb. 22, 2013, the entire content ofwhich is incorporated herein by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

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REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK

NOT APPLICABLE

BACKGROUND OF THE INVENTION

Cartilage tissue can be found throughout the human anatomy. The cellswithin cartilage tissue are called chondrocytes. These cells generateproteins, such as collagen, proteoglycan, and elastin, that are involvedin the formation and maintenance of the cartilage. Hyaline cartilage ispresent on certain bone surfaces, where it is commonly referred to asarticular cartilage. Articular cartilage contains significant amounts ofcollagen (about two-thirds of the dry weight of articular cartilage),and cross-linking of the collagen imparts a high material strength andfirmness to the tissue. These mechanical properties are important to theproper performance of the articular cartilage within the body.

Articular cartilage is not vascularized, and when damaged as a result oftrauma or degenerative causes, this tissue has little or no capacity forin vivo self-repair. A variety of therapeutic solutions have beenproposed for the treatment and repair of damaged or degeneratedcartilage. Tissue healing involves cell migration that redistributescells from the surrounding tissues to the injury site. In cartilagetissue, however, the ability of chondrocytes to migrate from theirnative lacunae site may be very limited due to the supposed rigidity ofthe matrix. To compensate for the migration deficiency, various surgicalinterventions for cartilage repair focus on delivering reparative cellsor tissues. For example, marrow stimulation attempts to tap marrow cellsby breaching the subchondral bone, although the mechanical durability ofresultant fibrocartilage is often unsatisfactory. Autologous chondrocyteimplantation (ACI) directly establishes a chondrocyte presence in thetreatment site through the delivery of culture-expanded chondrocytes.Despite being associated with some measure of clinical success, ACI isassociated with technical hurdles such as the cell culture preparation,two-stage surgical procedure, and challenging procedural aspects toplace small pieces of cartilage into the defect sites.

BRIEF SUMMARY OF THE INVENTION

In one aspect, mosaic cartilage compositions are provided. In someembodiments, the composition comprises:

-   -   a cartilage sheet comprising a plurality of interconnected        cartilage tiles; and    -   a biocompatible carrier.

In some embodiments, the composition comprises tiles that are separatedby channels having a depth that is less than the maximum thickness ofthe cartilage sheet. In some embodiments, the cartilage sheet has amaximum thickness of about 0.25 mm to about 5 mm. In some embodiments,the cartilage that is beneath the channels has a thickness of less than0.25 mm.

In some embodiments, the composition comprises tiles that are separatedby perforations. In some embodiments, the perforations aremicroperforations.

In some embodiments, the cartilage is articular cartilage. In someembodiments, the cartilage is non-decellularized cartilage. In someembodiments, the cartilage is from a human adult cadaveric donor age 15years or older. In some embodiments, the cartilage is from a humanjuvenile cadaveric donor.

In some embodiments, the tiles are circular in shape. In someembodiments, the tiles have an average diameter from about 0.5 mm toabout 3 mm. In some embodiments, the tiles are square or rectangular inshape. In some embodiments, the cartilage tiles have an average lengthand/or width from about 0.5 mm to about 3 mm. In some embodiments, thetiles are substantially uniform in size and/or shape.

In some embodiments, the cartilage sheet further comprises perforationsin the cartilage beneath one or more channels. In some embodiments, theperforations are microperforations.

In some embodiments, the biocompatible carrier comprises acryopreservation medium. In some embodiments, the cryopreservationmedium comprises dimethyl sulfoxide (DMSO) and serum.

In some embodiments, at least a portion of the cartilage sheet is coatedwith a biological adhesive. In some embodiments, the biological adhesiveis fibrin, fibrinogen, thrombin, fibrin glue, polysaccharide gel,cyanoacrylate glue, gelatin-resorcin-formalin adhesive, collagen gel,synthetic acrylate-based adhesive, cellulose-based adhesive, basementmembrane matrix, laminin, elastin, proteoglycans, autologous glue, or acombination thereof.

In some embodiments, at least a portion of the cartilage sheet iscombined with demineralized bone. In some embodiments, at least aportion of the cartilage sheet is combined with a bone or cartilagesubstrate that is seeded with stem cells.

In another aspect, methods of manufacturing a mosaic cartilagecomposition are provided. In some embodiments, the method comprises:

-   -   obtaining cartilage tissue from a human cadaveric donor;    -   cutting a plurality of channels or perforations into the        cartilage tissue, thereby forming a cartilage sheet comprising a        plurality of interconnected cartilage tiles that are separated        by the channels; and    -   suspending the cartilage sheet in a biocompatible medium.

In some embodiments, the cutting step comprises cutting a plurality ofchannels into the cartilage tissue, wherein each of the plurality ofchannels has a depth that is less than the maximum thickness of thecartilage tissue. In some embodiments, prior to the cutting step, thecartilage tissue has a maximum thickness of about 0.25 mm to about 5 mm.In some embodiments, the cartilage that is beneath the plurality ofchannels has a thickness of less than 0.25 mm.

In some embodiments, the cutting step comprises cutting a plurality ofperforations into the cartilage. In some embodiments, the perforationsare microperforations.

In some embodiments, the cartilage tissue is articular cartilage tissue.In some embodiments, the cartilage tissue is from a human adultcadaveric donor age 15 years or older. In some embodiments, thecartilage tissue is from a human juvenile cadaveric donor.

In some embodiments, the cutting step comprises cutting the cartilagetissue with a laser cutter, with a mechanical blade, or with amechanical press. In some embodiments, the cutting step comprisescutting the cartilage tissue with a laser cutter. In some embodiments,the cutting step comprising cutting the cartilage tissue with the lasercutter at a speed from about 15% to about 55%, a power from about 2% toabout 65%, and a frequency from about 200 Hz to about 2600 Hz.

In some embodiments, the cutting step comprises forming tiles that arecircular in shape. In some embodiments, the tiles have an averagediameter from about 0.5 mm to about 3 mm. In some embodiments, thecutting step comprises forming tiles that are square or rectangular inshape. In some embodiments, the tiles have an average length and/orwidth from about 0.5 mm to about 3 mm. In some embodiments, the cuttingstep comprises forming tiles that are substantially uniform in sizeand/or shape.

In some embodiments, the method further comprises making perforations incartilage beneath one or more of the channels. In some embodiments, theperforations are microperforations.

In some embodiments, following the cutting step, the method furthercomprises washing the cartilage sheet with a saline solution.

In some embodiments, the biocompatible carrier comprises acryopreservation medium. In some embodiments, the cryopreservationmedium comprises dimethyl sulfoxide (DMSO) and serum.

In some embodiments, prior to the suspending step, the method furthercomprises coating at least a portion of the cartilage sheet with abiological adhesive. In some embodiments, prior to the suspending step,the method further comprises combining at least a portion of thecartilage sheet with demineralized bone. In some embodiments, prior tothe suspending step, the method further comprises combining at least aportion of the cartilage sheet with a bone or cartilage substrate seededwith stem cells.

In another aspect, methods of treating a cartilage or bone defect in asubject are provided. In some embodiments, the method comprisesadministering to the subject a mosaic cartilage composition as describedherein.

In yet another aspect, methods of repairing cartilage in a subject areprovided. In some embodiments, the method comprises administering to thesubject a mosaic cartilage composition as described herein.

In still another aspect, kits (e.g., for treating a cartilage or bonedefect in a subject or for repairing cartilage in a subject) areprovided. In some embodiments, the kit comprises a mosaic cartilagecomposition as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Examples of cartilage constructs processed from cartilagetissue. (A) Cartilage can be cut into particles having a rectangularcolumnar shape. (B) Cartilage particles can be cut into particles havinga cylindrical or elliptical columnar shape. (C) Cartilage can be cutinto interconnected tiled or mosaic constructs. For example, thecartilage construct can have a width A, length B, and height C. Cuts,etches, or channels in the construct have a depth F and width H.Individual columns have a height F, width E, and length D. Subsequent tocutting, the construct has a minimum thickness G. (D) A cartilage tissuecan be cut, for example using a laser, on two or more sides (e.g., topand bottom).

FIG. 2. A standard curve for samples having known concentrations ofchondrocytes. The y-axis represents fluorescence readings from aCountess® automated cell counter, and the x-axis represents thechondrocyte concentration (cells/μl).

FIG. 3. Mean fluorescence readings for (A) chondrocyte samples fromadult donor A and (B) chondrocyte samples from juvenile donor B placedin six-well tissue culture plates.

FIG. 4. Mean fluorescence readings for chondrocyte samples from an adultdonor and from a juvenile donor, measured at day 1 and after culturingfor 6 weeks.

FIG. 5. Trypan Blue cell viability assay for Donors C, D, E, F, and G(also referred to as donors 1, 2, 3, 4, and 5, respectively). Cellviability was determined for laser cut and hand cut cartilage particles.The average cell viability is presented as a percentage. The term“Denovo” refers to a juvenile cartilage product that is hand cut into 1mm squares.

FIG. 6. Graph depicting the live cell count data for Trypan Blue andPresto Blue assays shown in the lower panel of FIG. 5.

FIG. 7. Trypan Blue cell viability assay at 6 weeks for laser cut andhand cut cartilage particles.

FIG. 8. Confocal microscope images depicting tissue edges (white arrow)of hand cut (A) and laser cut (B) cartilage pieces. InvitrogenLIVE/DEAD® stain was used on undigested cartilage sample for visualizingcells.

FIG. 9. Photographic images at 4× magnification of chondrocyte cellsgrowing out of hand cut (A) and laser cut (B) adult cartilage particles.Cartilage particles were placed in 12-well culture plates withchondrocyte growth medium containing 10% FBS and 2% antibiotic. Themedium was changed twice a week. The plates were cultured under standardcell culture conditions (37° C. incubator with 5% CO₂) and the imageswere obtained at 18 days.

FIG. 10. Schematic of an exemplary manufacturing method for cartilagecompositions.

FIG. 11. (A) An exemplary mosaic cartilage construct. The mosaiccartilage construct 1100 can include multiple tiles that are separatedby channels of a desired width or dimension. The tiles may be of anydesired shape (e.g., squares) and may have any desired dimension (e.g.,about 1 mm×1 mm square). The mosaic cartilage construct can also includeone or more apertures 1110. For example, apertures 1110 may representholes or passages that extend through the construct, from one side ofthe construct (e.g., a top surface) to an opposing side of the construct(e.g., a bottom surface). Apertures 1110 can be of any desired shape(e.g., square, circle, or oval), and may have any desired dimension(e.g., about 1 mm diameter circle, about 1 mm×1 mm square). (B)Apertures in the mosaic cartilage construct can operate to allow orenhance nutrient or liquid perfusion within and throughout the mosaiccartilage construct.

FIG. 12. An exemplary mosaic cartilage construct 1200 comprising amosaic pattern or circle-shaped tiles.

FIG. 13. Two exemplary mosaic cartilage constructs 1300 and 1310, eachcomprising a mosaic pattern of square-shaped tiles.

FIG. 14. A mosaic cartilage construct 1400 can be coated with abiological adhesive (e.g., fibrin gel or glue material), which can beused to secure the construct to a culture flask or well or to anapplication site. Cells can grow out of the cartilage construct, forexample out of a tile and into a channel disposed between two tiles.Similarly, cells can grow out of other construct surfaces, for examplebetween the cartilage and an application site (e.g., the surface of aflask, plate well, or patient treatment site).

FIG. 15. A juvenile mosaic cartilage construct at day 13, 10×magnification. Cells can be seen growing out of the mosaic cartilageedge and underneath the cartilage construct.

FIG. 16. A 4× magnification (A) and 10× magnification (B) of a mosaiccartilage construct. In both (A) and (B), cartilage cells can be seengrowing out of the cartilage construct, through the fibrin glue, andaway from the construct.

FIG. 17. (A) A cartilage particle having interlocking shapes. Thecartilage particle 1700 has multiple projections 1710. (B) Theprojections of the cartilage particle can interlock with the projectionsof a second cartilage particle having multiple projections, thusoperating to keep the particles together, or to inhibit the particlesfrom moving relative to one another, for example after being applied toa treatment site.

FIG. 18. A cartilage construct having multiple tiles separated byintervening channel sections. Discontinuous perforations can also beapplied to the channels (e.g., forming a dotted line down the channelsso as to provide a tear strip or perforated tear line feature). Thus, acartilage construct can include both channel sections and perforationsin the channel sections. Such perforations, which can extend through thethickness of the cartilage construct, can allow the migration ofnutrients and cells from an underlying bone or tissue to which thecartilage construct is applied.

FIG. 19. A cartilage construct having multiple tiles separated byintervening channel sections. Discontinuous apertures can be applied tothe channels (e.g., forming a dotted line down the channels). The openaperture ratio can be varied. For the construct depicted in FIG. 19, theopen aperture ratio is larger than that of the perforations shown inFIG. 18.

FIG. 20. Graph depicting the average live cell count data, as determinedby Presto Blue assays, for adult mosaic cartilage compositions after day1, week 3, week 6, week 9, and week 12 of culture.

FIG. 21. Comparison of total collagen content (mg/1 cc) in adult andjuvenile mosaic cartilage compositions after 12 weeks of culture.

FIG. 22. Sulfated glycosaminoglycan (sGAG) production in an adult mosaiccartilage composition after 12 weeks of culture.

FIG. 23. Immunohistochemistry for type II collagen in an adult mosaiccartilage composition after 12 weeks of culture. Brown staining in thecartilage indicates types II collagen produced by cells that grew out ofthe cartilage.

FIG. 24. Perforated mosaic cartilage composition produced by lasercutting.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

In one aspect, the present invention relates to mosaic cartilageconstructs comprising multiple interconnected cartilage tiles forcartilage and/or bone repair and regeneration. The mosaic cartilageconstructs disclosed herein provide a large surface area forfacilitating or promoting chondrocyte outgrowth from the cartilageconstruct into an application site (e.g., a site of bone or cartilageinjury), while the interconnectedness of the multiple tiles in theconstruct also promotes easier handling or surgical implantation ascompared to traditional autologous chondrocyte implantation (ACI) ormosaicplasty. Additionally, perforations in the cartilage facilitate thetransfusion of nutrients within, throughout, and across the cartilageconstruct, thereby enhancing chondrocyte outgrowth from the cartilageconstruct.

II. Definitions

As used herein, the term “interconnected tiles,” as used with referenceto a cartilage construct or sheet comprising multiple cartilage tiles,refers to cartilage tiles that are partially or incompletely connected,via cartilage tissue, to one or more other cartilage tiles within thesheet. As a non-limiting example, a sheet of cartilage into whichperforated lines have been cut, wherein the perforated lines separatethe cartilage into multiple smaller portions, or tiles, comprisescartilage tiles that are “partially connected.” In contrast, an uncutsheet of cartilage (e.g., a sheet of cartilage that has not beenperforated) is “completely connected.” In some embodiments,interconnected cartilage tiles are separated by discontinuousperforations (e.g., microperforations, apertures, bores, holes, or otherpassages), and a cartilage tile remains partially connected to one ormore other cartilage tiles via non-perforated cartilage adjacent to theperforated cartilage. In some embodiments, interconnected cartilagetiles are separated by channels that have a depth less than that of thedepth of the cartilage tiles, and a cartilage tile remains partiallyconnected to one or more other cartilage tiles via the cartilage beneaththe channels. In some embodiments, the interconnected cartilage tilesare all cut from a single piece of cartilage.

As used herein, the term “human adult donor” refers to a human donorthat is 15 years of age or older. In some embodiments, a human adultdonor is at least 18 years of age or older at the time of donation.

As used herein, the term “human juvenile donor” refers to a human donorthat is 12 years of age or younger. In some embodiments, a humanjuvenile donor is between the ages of 1 and 12 at the time of donation.

III. Cartilage Compositions

In one aspect, mosaic cartilage constructs are provided. In someembodiments, the composition comprises: a cartilage sheet comprising aplurality of interconnected tiles, wherein the tiles are separated bychannels having a depth that is less than the maximum thickness of thecartilage sheet; and a biocompatible carrier. In some embodiments, thecartilage sheet comprises a plurality of interconnected tiles areseparated by perforations. In some embodiments, the cartilage sheetcomprises a plurality of interconnected tiles are separated by channels,wherein the channels have a depth that is less than the maximumthickness of the cartilage sheet (e.g., less than the thickness of thetiles).

Cartilage Tile Formation and Separation

In some embodiments, the mosaic cartilage composition comprises acartilage sheet that has been cut into a plurality of interconnectedtiles. The interconnected cartilage tiles can be shaped as circles,squares, rectangles, triangles, ovals, polygons, zig-zags, irregularshapes, and the like, or other desired shape or combination of shapes.In some embodiments, the tiles are substantially uniform in size and/orshape. In some embodiments, the cartilage sheet comprises tile havingdifferent sizes and/or shapes. The interconnected tiles in the mosaicpattern hold together as a single unit, and also can operate tointerconnect or engage with tiles on other cartilage constructs whichmay also be applied to a treatment site.

In some embodiments, the mosaic cartilage construct comprises acartilage sheet of interconnected tiles having a circular shape. FIG. 12shows an exemplary cartilage construct 1200 having a circle mosaicpattern, wherein the circle-shaped tiles are separated by perforations.In some embodiments, the mosaic cartilage construct comprises acartilage sheet of interconnected tiles having a square and/orrectangular shape. FIG. 13 shows two exemplary cartilage constructs 1300each having a square mosaic pattern, wherein the square-shaped tiles areseparated by perforations.

In some embodiments, the cartilage sheet has a thickness of about 0.25mm to about 5 mm (e.g., about 0.25 mm, about 0.5 mm, about 0.75 mm,about 1 mm, about 1.5 mm, about 2 mm, about 2.5 mm, about 3 mm, about3.5 mm, about 4 mm, about 4.5 mm, or about 5 mm). In some embodiments,the cartilage sheet has a “maximum thickness,” e.g., the thickness ofthe cartilage tiles, and a “minimum thickness,” e.g., the thickness ofcartilage beneath one or more channels separating the cartilage tiles.In some embodiments, the cartilage sheet has a maximum thickness ofabout 0.25 mm to about 5 mm (e.g., about 0.25 mm, about 0.5 mm, about0.75 mm, about 1 mm, about 1.5 mm, about 2 mm, about 2.5 mm, about 3 mm,about 3.5 mm, about 4 mm, about 4.5 mm, or about 5 mm). In someembodiments, the cartilage sheet has a minimum thickness of less thanabout 0.25 mm, e.g., less than about 0.2 mm, about 0.15 mm, about 0.1mm, about 0.09 mm, about 0.08 mm, about 0.07 mm, about 0.06 mm, about0.05 mm, about 0.04 mm, about 0.03 mm, about 0.02 mm, or about 0.01 mm.

In some embodiments, the cartilage tiles have an average length and/oran average width from about 0.5 mm to about 3 mm (e.g., about 0.5 mm,about 0.6 mm, about 0.7 mm, about 0.8 mm, about 0.9 mm, about 1 mm,about 1.5 mm, about 2 mm, about 2.5 mm, or about 3 mm). In someembodiments, the cartilage tiles have an average diameter from about 0.5mm to about 3 mm (e.g., about 0.5 mm, about 0.6 mm, about 0.7 mm, about0.8 mm, about 0.9 mm, about 1 mm, about 1.5 mm, about 2 mm, about 2.5mm, or about 3 mm).

In some embodiments, the cartilage sheet has a length and/or a width ofabout 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm,about 10 mm, about 15 mm, about 20 mm, about 25 mm, about 30 mm, about35 mm, about 40 mm, about 45 mm, about 50 mm, about 60 mm, about 70 mm,about 80 mm, about 90 mm, or about 100 mm. In some embodiments, thecartilage sheet has a diameter of about 4 mm, about 5 mm, about 6 mm,about 7 mm, about 8 mm, about 9 mm, about 10 mm, about 15 mm, about 20mm, about 25 mm, about 30 mm, about 35 mm, about 40 mm, about 45 mm,about 50 mm, about 60 mm, about 70 mm, about 80 mm, about 90 mm, orabout 100 mm.

In some embodiments, the interconnected tiles are separated byperforations (e.g., microperforations, bores, apertures, and the like).An example of a mosaic cartilage composition comprising interconnectedtiles separated by perforations is shown in FIG. 24. In someembodiments, the perforations have an average diameter of about 5microns, about 10 microns, about 15 microns, about 20 microns, about 25microns, about 30 microns, about 35 microns, about 40 microns, about 45microns, about 50 microns, about 60 microns, about 70 microns, about 80microns, about 90 microns, or about 100 microns. Accordingly, in someembodiments, cartilage constructs are provided that have a porouscharacteristic. In some embodiments, the total cross-sectional area ofthe perforations (e.g., apertures) can be relatively small in comparisonto the total cross-sectional area of the cartilage construct itself. Insome embodiments, the aperture area ratio can be higher. Hence openaperture ratios can vary as desired. In some embodiments, the openaperture ratio can be within a range of 0% to 50% or higher. Constructswith greater open aperture ratios may operate to enhance the flow ofnutrients at the treatment site. In some embodiments, the open apertureratio is zero or substantially zero, such that the construct forms acontinuous or substantially continuous sheet. See, e.g., FIG. 18 andFIG. 19. The open aperture ratio depicted in FIG. 19 is larger than thatshown in FIG. 18.

In some embodiments, the interconnected tiles are separated by channels,wherein the channels have a depth that is less than the maximumthickness of the cartilage sheet (e.g., less than the thickness of thetiles). In some embodiments, the channels have a depth of about 0.2 mm,about 0.3 mm, about 0.4 mm, about 0.5 mm, about 0.6 mm, about 0.7 mm,about 0.8 mm, about 0.9 mm, about 1 mm, about 1.5 mm, about 2 mm, about2.5 mm, about 3 mm, about 3.5 mm, or about 4 mm. In some embodiments,wherein the cartilage sheet comprises channels separating the tiles, thecartilage sheet further comprises perforations in the cartilage beneathone or more of the channels.

In some embodiments, the channels or perforations (e.g.,microperforated) sections can present score features, such that a user(e.g., a physician or surgeon) can easily separate or break thecartilage construct into two or more pieces.

Cartilage Source

In some embodiments, the cartilage is articular cartilage. In someembodiments, the articular cartilage is obtained from an articularsurface of a joint (e.g., a knee joint or an elbow joint) or from a longbone (e.g., femur or tibia).

In some embodiments, the cartilage is from a human adult cadavericdonor. In some embodiments, the donor is an adult cadaveric donor thatis 18 years of age or older at the time of the donation. In someembodiments, the donor is an adult cadaveric donor that is between theages of 15 and 45 at the time of the donation. In some embodiments, thecartilage is from a human juvenile cadaveric donor. In some embodiments,the donor is a juvenile cadaveric donor that is between the ages of 3and 12 at the time of the donation.

Biocompatible Carrier

In some embodiments, the biocompatible carrier comprises a bufferedsolution. In some embodiments, the biocompatible carrier comprises acryopreservation medium. In some embodiments, the cryopreservationmedium comprises dimethyl sulfoxide (DMSO) and serum. In someembodiments, the biocompatible carrier comprises one or morecryoprotective agents such as, but not limited to, glycerol, DMSO,hydroxyethyl starch, polyethylene glycol, propanediol, ethylene glycol,butanediol, polyvinylpyrrolidone, or alginate.

In some embodiments, the biocompatible carrier comprises a growthmedium. Suitable examples of growth medium include, but are not limitedto, Dulbecco's Modified Eagle's Medium (DMEM) with 5% Fetal Bovine Serum(FBS). In some embodiments, growth medium includes a high glucose DMEM.In some embodiments, the biocompatible carrier (e.g., growth medium)comprises one or more antibiotics.

Additional Biological Components

In some embodiments, the mosaic cartilage composition is combined one ormore other biological components. For example, in some embodiments, atleast a portion of the mosaic cartilage composition is coated with abiological adhesive. See, e.g., FIG. 14. Suitable biological adhesivesinclude, but are not limited to, fibrin, fibrinogen, thrombin, fibringlue (e.g., TISSEEL), polysaccharide gel, cyanoacrylate glue,gelatin-resorcin-formalin adhesive, collagen gel, syntheticacrylate-based adhesive, cellulose-based adhesive, basement membranematrix (e.g., MATRIGEL®, BD Biosciences, San Jose, Calif.), laminin,elastin, proteoglycans, autologous glue, and combinations thereof.

In some embodiments, the mosaic cartilage composition is combined withdemineralized bone matrix. For example, in some embodiments the mosaiccartilage composition is combined with demineralized bone matrix at aratio of about 4:1, about 3:1, about 2:1, or about 1:1 demineralizedbone matrix:mosaic cartilage. Demineralized bone matrix can be prepared,e.g., by subjecting a bone substrate to acid, e.g., hydrochloric acid(HCl). Demineralized bone matrix is also commercially available.

In some embodiments, the mosaic cartilage composition is combined withcells such as stem cells. In some embodiments, the mosaic cartilagecomposition is combined with a bone or cartilage substrate that isseeded with stem cells. For example, in some embodiments, the mosaiccartilage composition is combined with a bone or cartilage substrate(e.g., cortical and/or cancellous bone substrate, demineralized corticaland/or cancellous bone substrate, an osteochondral substrate, or acartilage substrate) that is seeded with mesenchymal stem cells. Stemcell-seeded bone and cartilage substrates and methods of preparing suchsubstrates are described in published application US 2010/0124776 and inU.S. application Ser. No. 12/965,335, the contents of each of which areincorporated by reference herein.

In some embodiments, perforations (e.g., microperforations or apertures)can operate to facilitate the passage or infiltration ofcryopreservatives within and throughout the tissue construct. Forexample, a cryopreservation protocol may include using a laser to boreperforations (e.g., 100 micron diameter perforations or smaller) withina cartilage sheet (e.g., through channels, tiles, and other portions ofthe construct). Such perforations can allow a cryopreservative toquickly infiltrate otherwise solid tiles. Hence, the perforations cannot only allow for more cells to migrate out and for nutrients to flowfrom beneath or otherwise through, but can also allow for enhancedcryopreservation of cartilage constructs or sheets. Further, using thisapproach, large pieces of cartilage constructs can be cryopreserved, andperforation can operate to help maintain suitable temperature gradientsfor cryopreservation, as well as help facilitate a rapid infiltration ofcryopreservative into the cartilage, thus minimizing the duration ofcontact time between the cryopreservative and the cells until thecryopreservation state is reached. Thus, mosaic cartilage constructs ofthe present invention can be produced which have a long shelf life andwhich can be easily stored.

Quantifying Viable Chondrocytes and Characterizing CartilageCompositions

The mosaic cartilage compositions of the present invention can becharacterized with respect to the average number of chondrocytes in thetiles, the average chondrocyte viability in the tiles, or othercartilage characteristics or properties.

In some embodiments, the mosaic cartilage composition comprisescartilage tiles having an average chondrocyte viability of at least 50%,at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85% or higher. In some embodiments, the compositioncomprises cartilage tiles having at least about 50,000, at least about60,000, at least about 70,000, at least about 80,000, at least about90,000, at least about 100,000, at least about 150,000, at least about200,000, at least about 250,000, at least about 300,000, at least about350,000, at least about 400,000, at least about 450,000, at least about500,000, at least about 550,000, at least about 600,000, at least about650,000, at least about 700,000, at least about 750,000, at least about800,000, at least about 850,000, at least about 900,000, at least about950,000, or at least about 1 million viable chondrocytes per cubiccentimeter (cc). In some embodiments, the average chondrocyte viabilityor the amount of chondrocytes per cc is measured on day 1 following fromthe day of cutting.

The amount of chondrocytes in the cartilage tiles can be measured by anyof a number of cell counting assays. For example, in some embodiments, aTrypan Blue assay or a Presto Blue assay is used to quantify the numberof chondrocytes in the cartilage tiles. In some embodiments, thecartilage tissue is cut into the mosaic cartilage construct on day 0 andthen the amount of chondrocytes in the cartilage tiles of the mosaiccartilage construct is measured on day 1. In some embodiments, theamount of chondrocytes and/or cell viability is measured on day 1, day2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day12, day 13, or day 14 from the day of cutting. In some embodiments, theamount of chondrocytes and/or cell viability is measured 1 week, 2weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks from the day of cutting. Insome embodiments, for determining the amount of chondrocytes in asample, the sample is subjected to digestion, e.g., with collagenase, inorder to isolate chondrocytes for cell count and/or viability testing.

In some embodiments, a Trypan Blue assay is used to evaluate cell countand/or cell viability. The Trypan Blue assay is based upon the principlethat viable cells do not take up impermeable dyes such as Trypan Blue,but dead cells are permeable and take up the dye. Typically, Trypan Bluestain is added to a sample, then the sample is mixed. An aliquot of thesample is placed on a cell counter slide and the number of cells iscounted. The number of cells per cc is calculated based on the startingcartilage sample size.

In some embodiments, a Presto Blue assay is used to evaluate cell countand/or cell viability. The Presto Blue protocol involves an indirectchondrocyte cell count, using a metabolic assay. The cell count isperformed by using a standard curve of known concentrations ofchondrocytes to determine the count in the unknown samples. Typically, a1:10 ratio of PrestoBlue® reagent (Life Technologies, Carlsbad, Calif.)to cell culture medium is added to a sample so that the sample iscovered by the medium. The metabolic activity of the cells changes thecolor of the medium. After 3 hours incubation, 100 μl aliquots are takenfrom each sample and added to a multi-well plate for reading in a platereader.

In some embodiments, a cell counting technique other than the TrypanBlue assay or Presto Blue assay is used to determine chondrocyte cellcounts in a cartilage sample. For example, the LIVE/DEAD® stain (LifeTechnologies, Carlsbad, Calif.) or the CellTiter-Glo® Luminescent CellViability Assay (Promega, Madison, Wis.) can be used to evaluate cellviability. In some embodiments, a Quant-iT™ DNA Assay Kit (LifeTechnologies, Carlsbad, Calif.), such as with PicoGreen, can be used toassess DNA content, thereby determining cell count.

In some embodiments, cell viability can be calculated using thefollowing formula:(number of live cells/total number of live+dead cells)*100%=viabilitypercentage

The mosaic cartilage constructs can also be evaluated forcharacteristics of or chondrocyte outgrowth. For example, the mosaiccartilage constructs can be cultured for a period of time (e.g., 1, 2,3, 4, 5, or 6 weeks) and then assayed for one or more characteristics ofchondrocyte outgrowth, such as glycosaminoglycan production, thepresence of collagen, or the presence of one or more cartilage-specificbiomarkers. In some embodiments, the mosaic cartilage construct is froman adult donor, and the mosaic cartilage construct exhibits one or morecharacteristics of chondrocyte outgrowth, including but not limited toglycosaminoglycan production, collagen content, or cartilage-specificbiomarker expression, that is comparable to those characteristicsobtained from a mosaic cartilage construct from a juvenile donor andcultured under the same conditions.

In some embodiments, the mosaic cartilage construct exhibitsglycosaminoglycan (GAG) production after being cultured for a period oftime (e.g., as described herein in the Examples section). Chondrocytesfunction in part by producing GAGs and other components of thecartilaginous extracellular matrix. Hence, it is possible to evaluatethe chondrocyte activity of cartilage tissue by observingglycosaminoglycan production. The glycosaminoglycan content can bemeasured, for example, using a dimethylmethylene blue (DMMB) assay orusing Alcian Blue staining. In some embodiments, the levels of sulfatedGAGs (sGAGs) are measured. sGAGS are an important component of healthycartilage and can decrease with age and lead to the development ofosteoarthritis. sGAGs can be measured, for example, using a commerciallyavailable sGAG Assay Kit (Kamiya Biomedical Company, Seattle, Wash.).

In some embodiments, the mosaic cartilage construct exhibits collagenproduction after being cultured for a period of time (e.g., as describedherein in the Examples section). Collagen production and collagencontent can be measured, for example, using a hydroxyproline assay(BioVison, Milpitas, Calif.). Collagen production and collagen contentcan also be measured using an immunoassay (e.g., immunohistochemistry oran immunosorbent assay, e.g., ELISA assay), including but not limited toa Collagen Type II Antibody Staining Protocol.

IV. Methods of Manufacturing Cartilage Compositions

In another aspect, methods of manufacturing cartilage compositions areprovided. In some embodiments, the method comprises:

-   -   obtaining cartilage tissue from a human cadaveric donor;    -   cutting a plurality of channels or perforations into the        cartilage tissue, thereby forming a cartilage sheet comprising a        plurality of interconnected tiles that are separated by the        channels; and    -   suspending the cartilage sheet in a biocompatible medium.

In some embodiments, the cartilage tissue is harvested from an adultcadaveric donor. In some embodiments, the cartilage tissue is harvestedfrom an adult cadaveric donor that is between the ages of 15 and 45 atthe time of the donation. In some embodiments, the cartilage tissue isharvested from a juvenile cadaveric donor. Tissue can be harvested fromany cartilaginous region of the cadaveric donor. In some embodiments,cartilage is harvested from the knee joint of the donor or from a longbone. In some embodiments, articular cartilage is harvested from thedonor. In some embodiments, the cartilage that is obtained from thedonor is sliced to a thickness of about 0.25 mm to about 5 mm (e.g.,about 0.25 mm, about 0.5 mm, about 0.75 mm, about 1 mm, about 1.5 mm,about 2 mm, about 2.5 mm, about 3 mm, about 3.5 mm, about 4 mm, about4.5 mm, or about 5 mm, or from about 0.5 mm to about 2 mm) before thecutting step.

The interconnected cartilage tiles can be cut into circles, squares,rectangles, triangles, ovals, polygons, zig-zags, irregular shapes, andthe like, or other desired shape or combination of shapes. In someembodiments, the tiles are substantially uniform in size and/or shape.In some embodiments, the cartilage sheet comprises tile having differentsizes and/or shapes.

In some embodiments, the cutting step comprises cutting a plurality ofchannels into the cartilage tissue, wherein each of the plurality ofchannels has a depth that is less than the maximum thickness of thecartilage tissue. In some embodiments, the channels have a depth that isless than the maximum thickness of the cartilage sheet (e.g., less thanthe thickness of the cartilage tissue and/or the thickness of thetiles). In some embodiments, the channels have a depth of about 0.2 mm,about 0.3 mm, about 0.4 mm, about 0.5 mm, about 0.6 mm, about 0.7 mm,about 0.8 mm, about 0.9 mm, about 1 mm, about 1.5 mm, about 2 mm, about2.5 mm, about 3 mm, about 3.5 mm, or about 4 mm. In some embodiments,the cartilage that is beneath the channels has a thickness of less thanabout 0.25 mm, e.g., less than about 0.2 mm, about 0.15 mm, about 0.1mm, about 0.09 mm, about 0.08 mm, about 0.07 mm, about 0.06 mm, about0.05 mm, about 0.04 mm, about 0.03 mm, about 0.02 mm, or about 0.01 mm.

In some embodiments, the cutting step comprises cutting a plurality ofperforations into the cartilage. Perforations can include, for example,microperforations, bores, apertures, and the like). In some embodiments,the perforations are microperforations. In some embodiments, theperforations are apertures. In some embodiments, perforations may be onthe order of tens of microns in dimension, or less. In some embodiments,perforations may be on the order of millimeters in dimension, or less.For example, in some embodiments, the perforations have an averagediameter of about 5 microns, about 10 microns, about 15 microns, about20 microns, about 25 microns, about 30 microns, about 35 microns, about40 microns, about 45 microns, about 50 microns, about 60 microns, about70 microns, about 80 microns, about 90 microns, or about 100 microns.

In some embodiments, the cutting step comprises cutting a plurality ofchannels into the cartilage tissue, and further comprises cuttingperforations in the cartilage beneath one or more of the channels.

In some embodiments, the cartilage tissue is cut into tiles having anaverage length and/or an average width from about 0.5 mm to about 3 mm(e.g., about 0.5 mm, about 0.6 mm, about 0.7 mm, about 0.8 mm, about 0.9mm, about 1 mm, about 1.5 mm, about 2 mm, about 2.5 mm, or about 3 mm).In some embodiments, the cartilage tissue is cut into tiles having anaverage diameter from about 0.5 mm to about 3 mm (e.g., about 0.5 mm,about 0.6 mm, about 0.7 mm, about 0.8 mm, about 0.9 mm, about 1 mm,about 1.5 mm, about 2 mm, about 2.5 mm, or about 3 mm).

In some embodiments, the cartilage tissue is cut into a sheet having alength and/or a width of about 4 mm, about 5 mm, about 6 mm, about 7 mm,about 8 mm, about 9 mm, about 10 mm, about 15 mm, about 20 mm, about 25mm, about 30 mm, about 35 mm, about 40 mm, about 45 mm, about 50 mm,about 60 mm, about 70 mm, about 80 mm, about 90 mm, or about 100 mm. Insome embodiments, the cartilage tissue is cut into a sheet having adiameter of about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm,about 9 mm, about 10 mm, about 15 mm, about 20 mm, about 25 mm, about 30mm, about 35 mm, about 40 mm, about 45 mm, about 50 mm, about 60 mm,about 70 mm, about 80 mm, about 90 mm, or about 100 mm.

In some embodiments, the cartilage tissue is cut by hand. In someembodiments, the cartilage tissue is cut using a cutting mechanism. Insome embodiments, the cutting mechanism is a laser cutting apparatus, amechanical blade, a manual cutting apparatus, a manual pressingapparatus, or the like. In some embodiments, the cutting mechanismcomprises a pneumatic press, such as an air press or an oil press, or ascrew press.

In some embodiments, the cartilage tissue is cut using a laser cuttingapparatus. For example, in some embodiments, the laser cutting apparatusis a laser engraver. Non-limiting examples of suitable engraving lasersinclude CO₂ engraving lasers, such as the Epilog Zing 30 Watt CO₂engraving laser. In some embodiments, the cutting step comprises cuttingthe cartilage tissue with the laser cutting apparatus at a speed fromabout 10% to about 55% (e.g., about 10%, about 15%, about 20%, about25%, about 30%, about 35%, about 40%, about 45%, about 50%, or about55%), a power from about 0% to about 65% (e.g., about 0%, about 1%,about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%,or about 65%), and a frequency from about 10 Hz to about 2600 Hz (e.g.,about 10 Hz, about 20 Hz, about 30 Hz, about 40 Hz, about 50 Hz, about60 Hz, about 70 Hz, about 80 Hz, about 90 Hz, about 100 Hz, about 150Hz, about 200 Hz, about 250 Hz, about 300 Hz, about 350 Hz, about 400Hz, about 450 Hz, about 500 Hz, about 550 Hz, about 600 Hz, about 650Hz, about 700 Hz, about 750 Hz, about 800 Hz, about 850 Hz, about 900Hz, about 950 Hz, about 1000 Hz, about 1100 Hz, about 1200 Hz, about1300 Hz, about 1400 Hz, about 1500 Hz, about 1600 Hz, about 1700 Hz,about 1800 Hz, about 1900 Hz, about 2000 Hz, about 2100 Hz, about 2200Hz, about 2300 Hz, about 2400 Hz, about 2500 Hz, or about 2600 Hz). Insome embodiments, the cutting step comprising cutting the cartilagetissue with the laser cutter at a speed from about 10% to about 50%, apower from about 0% to about 45%, and a frequency from about 10 Hz toabout 2400 Hz. In some embodiments, the cutting step comprising cuttingthe cartilage tissue with the laser cutter at a speed from about 15% toabout 55%, a power from about 2% to about 65%, and a frequency fromabout 200 Hz to about 2600 Hz. Suitable speeds, powers, and frequenciesfor cutting the cartilage tissue are shown in Table 1.

In some embodiments, on average at least 50% of the chondrocytes in thecartilage composition are viable. In some embodiments, an average atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85% or more of the chondrocytes in thecartilage composition are viable. In some embodiments, the cartilageparticles comprise at least about 50,000, at least about 60,000, atleast about 70,000, at least about 80,000, at least about 90,000, atleast about 100,000, at least about 150,000, at least about 200,000, atleast about 250,000, at least about 300,000, at least about 350,000, atleast about 400,000, at least about 450,000, at least about 500,000, atleast about 550,000, at least about 600,000, at least about 650,000, atleast about 700,000, at least about 750,000, at least about 800,000, atleast about 850,000, at least about 900,000, at least about 950,000, orat least about 1 million viable chondrocytes per cubic centimeter (cc).In some embodiments, the average chondrocyte viability or the amount ofchondrocytes per cc is measured on day 1 following from the day ofcutting. The amount of chondrocytes and/or number of viable chondrocytesin a cartilage sample can be measured as described herein, for exampleas described in Section III above.

Further Processing Steps

In some embodiments, following the cutting step, the mosaic cartilagecomposition is not subjected to an additional processing step prior tosuspending the cartilage composition in the biocompatible carrier. Insome embodiments, following the cutting step, the mosaic cartilagecomposition can be subjected to one or more additional processing stepsprior to suspending the cartilage composition in the biocompatiblecarrier. In some embodiments, the cartilage composition is washed with asaline solution. In some embodiments, the cartilage composition istreated with one or more enzymes that promote the release of chondrocytecells from cartilage matrix. For example, collagenase can be applied tohelp release chondrocyte cells from the cartilage matrix of thecomposition. In some embodiments, the cartilage composition is mixedwith collagenase and/or pronase and incubated in a growth medium such asDulbecco's Modified Eagle's Medium (DMEM) for a suitable length of timefor releasing the chondrocytes.

In some embodiments, the cartilage composition is combined withdemineralized bone matrix. For example, in some embodiments thecartilage composition is combined with demineralized bone matrix at aratio of about 4:1, about 3:1, about 2:1, or about 1:1 demineralizedbone matrix:cartilage composition). Demineralized bone matrix can beprepared, e.g., by subjecting a bone substrate to acid, e.g.,hydrochloric acid (HCl). Demineralized bone matrix is also commerciallyavailable.

In some embodiments, the cartilage composition is combined with cellssuch as stem cells. In some embodiments, the cartilage composition iscombined with a bone or cartilage substrate that is seeded with stemcells. For example, in some embodiments, the cartilage composition iscombined with a bone or cartilage substrate (e.g., cortical and/orcancellous bone substrate, demineralized cortical and/or cancellous bonesubstrate, an osteochondral substrate, or a cartilage substrate) that isseeded with mesenchymal stem cells. Stem cell-seeded bone and cartilagesubstrates and methods of preparing such substrates are described inU.S. 2010/0124776 and U.S. application Ser. No. 12/965,335, the contentsof each of which are incorporated by reference herein.

In some embodiments, at least a portion of the cartilage composition iscombined with a biological adhesive. Suitable biological adhesivesinclude, but are not limited to, fibrin, fibrinogen, thrombin, fibringlue (e.g., TISSEEL), polysaccharide gel, cyanoacrylate glue,gelatin-resorcin-formalin adhesive, collagen gel, syntheticacrylate-based adhesive, cellulose-based adhesive, MATRIGEL® (BDBiosciences, San Jose, Calif.), laminin, elastin, proteoglycans, andcombinations thereof.

In some embodiments, the cartilage composition is suspended in abiocompatible carrier. In some embodiments, the biocompatible carriercomprises a buffered solution (e.g., an aqueous buffered solution). Insome embodiments, the biocompatible carrier comprises a cryopreservationmedium. In some embodiments, the cryopreservation medium comprisesdimethyl sulfoxide (DMSO) and serum. In some embodiments, thebiocompatible carrier comprises one or more cryoprotective agents suchas, but not limited to, glycerol, DMSO, hydroxyethyl starch,polyethylene glycol, propanediol, ethylene glycol, butanediol, orpolyvinylpyrrolidone.

V. Therapeutic Uses of Cartilage Compositions

The mosaic cartilage compositions described herein can be used to treatsubjects in need thereof. Without being bound to a particular theory, itis believed that the methods of cutting cartilage described herein canfacilitate or enhance the migration of cells out of the cartilage. Forexample, the cutting of channels and/or perforations onto the surface ofcartilage tissue to form interconnected tiles creates a construct havingincreased surface area relative to a cartilage tissue lacking saidchannels. This increased surface area can increase or enhance themigration of chondrocytes out of the cartilage tissue relative to theamount of chondrocyte migration in an uncut cartilage tissue.Additionally, the channels and/or perforations can operate to allownutrients to transfuse easily within, throughout, and across thecartilage construct at an injury site, and thus may contribute toenhanced regeneration and healing.

When the mosaic cartilage compositions as described herein areadministered to a subject, the chondrocytes within the cartilage sheetcan migrate out of the sheet and carry out repair and regenerationfunctions. For example, the chondrocytes can reproduce and form newcartilage via chondrogenesis. In this way, a mosaic cartilagecomposition which is applied to a site within a patient can be used totreat cartilage and/or bone defects. For example, chondrocytes from themosaic cartilage composition can reproduce and generate new cartilage insitu. The newly established chondrocyte population and cartilage tissuecan fill defects, and integrate with existing native cartilage and/orsubchondral bone at the treatment site.

In some embodiments, the mosaic cartilage compositions described herein(e.g., a composition comprising a cartilage sheet comprising a pluralityof interconnected tiles and a biocompatible carrier) are administered toa subject having a bone or cartilage defect. In some embodiments, themosaic cartilage composition is administered at a site of defect incartilage, bone, ligament, tendon, meniscus, joint, or muscle. In someembodiments, the subject has a degenerative defect or injury. In someembodiments, the subject has a traumatic defect or injury. In someembodiments, the subject has osteoarthritis. In some embodiments, thesubject has a muscle defect.

In some embodiments, the mosaic cartilage compositions described hereinare administered to a subject to repair cartilage or promote cartilagegrowth or regeneration in the subject. In some embodiments, the mosaiccartilage composition is administered to a joint (e.g., knee joint), tobone (e.g., femur or humerus), or to cartilage.

In some embodiments, the mosaic cartilage compositions described hereinare administered to a subject having soft tissue defects, for the repairand regeneration thereof. In some embodiments, the composition isadministered to a ligament, tendon, or muscle. In some embodiments, thesoft tissue defect is a sprain, strain, contusion, or stress injury to aligament, tendon, or muscle.

In some embodiments, a mosaic cartilage composition as described hereinis administered locally to the subject. In some embodiments, thecomposition is surgically implanted in the subject. In some embodiments,the composition is administered in a minimally invasive procedure, e.g.,arthroscopy.

In some embodiments, a mosaic cartilage composition as described hereinis provided to a user (e.g., a physician or a surgeon) as a larger sheetand can be broken into smaller pieces by the user to an appropriate sizeto suit the size of the defect (e.g., cartilage defect) being treated.For example, a cartilage construct can broken or separated into twopieces along one or more channels of the construct, or along aperforated section of the construct.

VI. Kits

In still another aspect, kits comprising a mosaic cartilage compositionas described herein are provided. In some embodiments, the kit comprisesa mosaic cartilage composition comprising a cartilage sheet comprising aplurality of interconnected tiles; and a biocompatible carrier.

In some embodiments, the kits are used for treating a subject having adefect in cartilage, bone, ligament, tendon, meniscus, joint, or muscle.In some embodiments, the kits are used for treating a subject having adegenerative defect or injury cartilage, bone, ligament, tendon,meniscus, joint, or muscle; a subject having a traumatic defect orinjury cartilage, bone, ligament, tendon, meniscus, joint, or muscle; ora subject having osteoarthritis.

In some embodiments, a kit comprises a mosaic cartilage composition asdescribed herein packaged in a container for storage and/or shipment. Insome embodiments, the kit further comprises instructions foradministering the composition.

In some embodiments, a kit comprises a mosaic cartilage compositioncomprising a cartilage sheet comprising a plurality of interconnectedtiles as described herein, optionally along with biological adhesivecomponents (e.g., fibrinogen and thrombin, for a fibrin glue). In someembodiments, cartilage particles and biological adhesive (e.g., fibringlue) components are packaged separately, and a surgeon or user adds thefibrin glue to the surgery site prior to placement of the cartilage. Insome embodiments, the biological adhesive (e.g., fibrin glue) iscombined with the cartilage composition prior to administration at thetreatment site.

In some instances, a kit comprises the packaged cartilage compositionwith bone and/or stem cell components. For example, in some embodiments,a kit comprises a cartilage composition with demineralized bone matrix.In some embodiments, a kit comprises a cartilage composition with cells(e.g., stem cells). In some embodiments, a kit comprises a cartilagecomposition with a bone or cartilage substrate seeded with cells (e.g.,adipose derived mesenchymal adult stem cells combined with a bonesubstrate, as described in U.S. 2010/0124776, or adipose derivedmesenchymal adult stem cells combined with an osteochondral or cartilagesubstrate, as described in U.S. application Ser. No. 12/965,335).

VII. Examples

The following examples are offered to illustrate, but not to limit, theclaimed invention.

Example 1 Laser Cutting to Generate Mosaic Cartilage Sheets

Laser cutting techniques can provide a cost effective approach for thepreparation of interconnected mosaic cartilage sheets withoutsacrificing cell viability. As described below, mosaic cartilage sheetscomprising a plurality of interconnected tiles as prepared by laserprocessing techniques showed cell viability results that were comparableto the cell viability results observed when using manual cuttingtechniques. The use of laser cutting techniques reduces cost,contamination, and processing time, and additionally allows forincreased amounts of donor tissue product to be utilized.

Tissue cutting experiments were performed using an Epilog Zing 30 WattCO2 engraving laser on sheets of juvenile or adult cartilage to formminced cartilage particles and mosaic cartilage constructs. Table 1shows the results of the tissue cutting experiments at varying speeds,powers, and frequencies.

TABLE 1 Laser Settings A. Low Range Settings Test: 2 mm square patterncut, 1 mm thick samples used Laser Settings Speed (%) Power (%)Frequency (Hz) Result/outcome: 30 10 1350 Etches tissue, no burning,doesn't cut entirely through (mosaic) 30 10 1000 Etches tissue, noburning, doesn't cut entirely through (mosaic) 30 10 750 Etches tissue,no burning, doesn't cut entirely through (mosaic) 30 8 750 Some browningof tissue, perforations through tissue 25 8 750 Completely cut throughtissue, some brown edges 25 8 650 Completely cut through tissue, somebrown edges 25 8 400 Completely cut through tissue, no browning 25 5 400Etched tissue, some browning, does not cut entirely through 25 5 300Etched tissue, no browning, does not cut entirely through 20 5 300Etched tissue, no browning, nearly complete full thickness cut 20 2 300Etched tissue, no browning, does not cut entirely through 20 0 300Etched tissue, no browning, etching not very deep 20 2 200 Etchedtissue, no browning, nearly complete full thickness cut 20 2 100 Etchedtissue, no browning, nearly complete full thickness cut 20 2 50 Etchedtissue, no browning, nearly complete full thickness cut 20 2 25 Etchedtissue, no browning, nearly complete full thickness cut withperforations through tissue 20 2 10 Perforations (full thickness) onlythrough tissue no complete etched line 20 1 10 Perforations only, not afull thickness cut 20 0 10 Perforations only, not a full thickness cut10 0 10 Laser very slow moving, tissue etched with perforations (fullthickness), no solid line cut B. High Range Settings Test: 2 mm squarepattern cut, 1 mm thick samples used Laser Settings Speed (%) Power (%)Frequency (Hz) Result/outcome: 30 30 2000 Some browning of edges,complete cut full thickness cut 35 30 2000 Less browning than abovesettings, complete full thickness cut 35 35 2000 Some browning of edges,complete cut full thickness cut 35 35 2200 Some browning of edges,complete cut full thickness cut 35 40 2200 Some browning of edges,complete cut full thickness cut 35 40 2400 Browning of edges, completefull thickness cut 35 45 2400 dark brown edges, complete cut through

Based at least in part upon these findings, it was determined that lasersettings at 15-55% speed, 2-65% power, and 200-2600 Hz frequency providedesirable results for producing interconnected mosaic cartilageconstructs.

Example 2 Characterization of Articular Cartilage from Adult or JuvenileDonors

Fresh cadaveric adult and juvenile articular cartilage tissue sampleswere processed into minced cartilage particles using either a lasercutting protocol or a hand cutting protocol. The adult donors werebetween fifteen and thirty six years of age, and the juvenile donorswere between the ages of three months and 12 years. For the lasercutting method, the cartilage was shaved into thin slices (e.g., sheetshaving a thickness of 1-5 mm) using a scalpel, and the sliced sheetswere minced into small particles (e.g., 1 mm, 2 mm, and/or 3 mmparticles) using an Epilog Zing 30 Watt engraving laser. The lasercutting pattern was designed with a CorelDRAW® graphics softwareprogram. The cartilage was minced into square shaped particles, usingenergy levels and other laser parameters as described in Table 1. Duringthe laser cutting procedure, the cartilage was maintained in a hydratedstate. The minced particles were then washed with a phosphate bufferedsaline (PBS) solution. Cartilage particles were characterized for cellcount, cell viability, and chondrocyte growth as described below. Mosaiccartilage compositions can also be prepared, e.g., into 3×3 sheets of 2mm cartilage tiles as described in Example 3 below, and characterizedfor cell count, cell viability, and chondrocyte growth.

Using samples having known concentrations of chondrocytes, a standardcurve was prepared as shown in FIG. 2. The y-axis representsfluorescence readings from a Countess® automated cell counter, and thex-axis represents the chondrocyte concentration (cells/μl).

Cell Counting, Donors A (Adult) and B (Juvenile), Day One:

Some of the harvested chondrocytes were tested for cell count on the dayof mincing (day 1) using a Trypan blue staining protocol followed byanalysis in a Countess® automated cell counter. Cartilage particles weredigested with collagenase to isolate chondrocytes, and that mixture wasthen filtered through a 105 micron filter to separate any undigestedmatrix from the isolated cells. For the experiments illustrated by FIGS.3A and 3B, equal amounts of chondrocyte samples were placed in theindividual plate wells for evaluation.

As depicted in FIG. 3A, adult donor cartilage tissue that was mincedwith laser cutting provided a mean fluorescence reading of 21,636 (Std.Dev. 578; CV % 2.67), which corresponds to a cell count of 42,622chondrocytes/μl, using the standard curve of FIG. 2. The adult donorcartilage tissue that was minced with hand cutting provided a meanfluorescence reading of 24,853 (Std. Dev. 1507; CV % 6.06), whichcorresponds to a cell count of 52,642 chondrocytes/μl. As depicted inFIG. 3B, juvenile donor cartilage tissue that was minced with lasercutting provided a mean fluorescence reading of 27,528 (Std. Dev. 2494;CV % 9.06), which corresponds to a cell count of 60,974 chondrocytes/μl.The juvenile donor cartilage tissue that was minced with hand cuttingprovided a mean fluorescence reading of 41,088 (Std. Dev. 3472; CV %8.45), which corresponds to a cell count of 103,211 chondrocytes/μl.Based on these results, it was observed that in terms of cell count,there may be no large differences between the laser cutting and handcutting methods.

FIG. 4 shows mean fluorescence readings as described above. The numberswere calculated using a standard curve and the fluorescence reading froma Presto Blue metabolic assay when evaluated in the plate reader. Sixweek cell counts were also performed using a Presto Blue assay.

Cell Counting, Donors C to G (Six Week):

To compare how chondrocytes from both adult and juvenile cartilage growout of the cartilage matrix, a 6-week explant study was conducted. Threeresearch-consented adult donors (donors C, E, and G) and tworesearch-consented juvenile donors (donors D and F) were obtained.Samples were cut into sheets approximately 1 mm thick and minced by handor laser cut into 2 mm cubes and measured into 0.3 ml aliquots.Cartilage particles were placed into plate wells along with TISSEELfibrin glue (Baxter, Deerfield, Ill.), which provided a support fromwhich the chondrocytes could grow out of the cartilage samples. Nocollagenase was used on the cells. Chondrocyte media (Cell Applications,San Diego, Calif.) was then added and changed twice weekly.

Cell counting was conducted after six weeks using either (A) a TrypanBlue staining protocol followed by analysis in a Countess® automatedcell counter, or (B) a Presto Blue staining protocol followed byanalysis in a Synergy™ H1 hybrid plate reader. The Presto Blue protocolinvolves an indirect chondrocyte cell count, using a metabolic assay.The cell count is performed by using a standard curve of knownconcentrations of chondrocytes to determine the count in the unknownsamples. Typically, where the chondrocytes are combined with fibrin, ametabolic assay and hybrid reader can be used to indirectly determinethe chondrocyte cell count, by evaluating the metabolic activity. Here,it may be assumed that a majority of the cells (e.g., 95% to 98% ormore) are viable.

FIG. 5 shows the live cell number count and viability results for theTrypan Blue protocol, and the live cell count number results for thePresto Blue protocol. As depicted in the Trypan Blue live cell testresults, there were 1,052,167±989,536 of live cells per cc of freshcartilage using laser cutting, and 375,333±295,846 live cells per cc offresh cartilage using hand cutting.

FIG. 6 shows the live cell count number results for the Trypan Blue andPresto Blue protocols, and is based on cell count data shown in FIG. 5.With regard to the Trypan Blue and Presto Blue cell count results shownhere, a single ANOVA analysis was performed and there was no significantdifference using these two methods regarding live cell number.

Cell Counting, Donors C to G:

FIG. 7 shows day 1 (i.e., one day after cutting) cell viability assayfor Donors C to G using the Trypan Blue protocol, which are based on theviability % results depicted in FIG. 5. As depicted here, the averagecell viability is about 86% for both laser cut cartilage and hand cutcartilage. Hence, it was observed that cartilage tissue can be mincedwith laser cutting, without sacrificing cell viability relative to handcutting methods. With regard to the Trypan Blue viability results shownin FIG. 7, a single ANOVA analysis was performed and there was nosignificant difference using these two methods regarding cell viability.

FIGS. 8A and 8B are confocal microscope images depicting tissue edges(white arrow) of hand cut and laser cut (respectively) cartilage pieces.These results indicate that there was not a significant difference ofcell viability when comparing laser cut and hand cut cartilage tissuesamples. For this study, LIVE/DEAD® stain (Life Technologies, Carlsbad,Calif.) was used. Briefly, undigested cartilage particles were placed inwells of a 24-well plate. 1 ml PBS was added to each well and 0.5 μl ofthe red and green dye was then added. The plates were covered with foiland allowed to sit for a minimum of 15 minutes. The cartilage particleswere then placed on slides and the images captured by confocalmicroscopy on the laser setting.

It was also observed that laser cutting could be accomplished morequickly than hand cutting. For example, an equivalent amount of tissuecould be minced in 8 hours via manual cutting, versus 0.5 hours vialaser cutting. Moreover, it was observed that it was easier to obtainuniformly shaped tissue pieces using laser cutting, as compared withhand cutting.

Microscopy Observations at Eighteen Days:

FIGS. 9A and 9B provide photographic images of chondrocyte cells growingout of hand cut (FIG. 9A) and laser cut (FIG. 9B) adult cartilageparticles. Specifically, cartilage was obtained from an adult donor, andminced with either laser cutting or manual cutting protocols. The mincedcartilage particles were placed in 12 well culture plates, usingchondrocyte growth medium with 10% FBS and 2% antibiotic. The media waschanged twice a week. The plates were cultured in a 37° C. incubatorwith 5% CO₂ (e.g., standard cell culture conditions). The images (4×magnification) were obtained at 18 days. As shown here, chondrocyteswere observed to grow out of the minced particles.

It has previously been suggested that adult cartilage is not well suitedfor use in allogeneic grafts. However, this example demonstrates thatadult cartilage constructs, when cultured for a period of time, exhibitcomparable chondrocyte outgrowth and matrix production as juvenilecartilage constructs. Thus, cartilage constructs derived from humanadult donors or from human juvenile donors can be useful for repairingcartilage defects in subjects in need thereof.

Example 3 12-Week Explant Study to Characterize Cartilage Samples

To further compare chondrocyte outgrowth and matrix production of mosaiccartilage compositions, a 12-week explant study was performed. Threeresearch consented adult donors and two research consented juveniledonors were obtained. For cell counting, a 1:10 ratio of PrestoBlue®(Life Technologies, Carlsbad, Calif.) to media was used. Collagen typeII immunohistochemistry was performed on samples after the 12 week timepoint, as well as sulfated glycosaminoglycans (sGAG) assay (KamiyaBiomedical Company, Seattle, Wash.), hydroxyproline assay (BioVison,Milpitas, Calif.), and DNA analysis with a Pico Green Assay (Invitrogen,Grand Island, N.Y.). All outcome measures were evaluated using singleANOVA analysis. Significance was considered as p≦0.05.

Cartilage Explant Protocol:

1. Remove media and wash cartilage samples with PBS+2% antibiotic.

2. Place tissue on chilled laser plate as flat as possible.

3. Add several drops of PBS+2% antibiotic to tissue to keep moist duringcutting, place plate inside laser.

4. Select desired laser template and set laser settings to Speed=50%,Power=30%, and Hz=2000. Select print and press go on the laser.

5. Measure out 0.3 cc of cartilage using 15 ml conical tube with 2.7 mLof medium and bring up to 3 mL. Mosaic cartilage sheets comprising 2 mmsquares in a 3×3 (6 mm×6 mm) sheet were formed.

6. Place TISSEL fibrin glue in base of well and add cartilage.

7. After all samples have been added to plate, allow 30 minutes for glueto dry.

8. After cartilage has attached and glue has dried, add 4 mL mediumgently to well as to not disturb the cartilage.

9. On day 1, remove medium to run Presto Blue, 1 mL medium+110 μL PrestoBlue into each well and incubate for 4.5 hours. Remove medium and PrestoBlue and rinse with PBS+2% antibiotic. Replace medium.

10. Change medium twice weekly.

11. Incubate plates. Check for cell explant growth with Presto Blue atweeks 3, 6, 9, and 12.

TABLE 2 Summary of samples Laser cut minced Laser cut mosaic Assay withfibrin glue with fibrin glue Cell count n = 3 n = 3 (Presto Blue) PrestoBlue DNA n = 3 n = 3 assay (cell count) GAG Analysis n = 2 n = 2

TABLE 3 12 well plate layout (sample designation) Laser Cut minced LaserCut minced Mosaic Cut Sample: Mosaic Cut Sample: sample: GAG sample:Presto GAG Analysis, Presto Presto Blue/histology Analysis, Presto BlueBlue/histology Blue Laser Cut minced Laser Cut minced Mosaic Cut Sample:Mosaic Cut Sample: sample: GAG sample: Spare in case GAG Analysis,Presto Spare in case of Analysis, Presto Blue of contamination Bluecontamination Laser Cut minced EMPTY well or back- Mosaic Cut Sample:EMPTY well or back- sample: GAG up well if enough tissue GAG Analysis,Presto up well if enough Analysis, Presto Blue Blue tissueResults

The 12-week study demonstrated that mosaic cartilage compositionsexhibited cell outgrowth and matrix production. Mosaic cartilagecompositions from adult donors exhibited an increase in the number ofcells per sheet/well over the course of the 12-week explant study (FIG.20), as did mosaic cartilage compositions from juvenile donors (data notshown).

A hydroxyproline assay was used to determine the total collagen contentof the explants. As shown in Table 4 and FIG. 21, mosaic cartilagecompositions from adult donors had a total collagen content of57.14±0.72 mg/ml. Juvenile donors had a total of 44.95±3.32 mg/ml,resulting in no statistical difference.

TABLE 4 Results for hydroxyproline and sGAG after 12 weeks of explantAverage Result Standard Deviation P- Statistically Assay Adult JuvenileAdult Juvenile value Different? Hydroxyproline 57.13766 44.951860.716883 3.324416 0.9 NO (ug/well) sGAG (ug/well) 199541 197442.637371.07 15857.74 NO

Sulfated glycosaminoglycans (sGAGS) are an important component ofhealthy cartilage and can decrease with age and lead to the developmentof osteoarthritis. Average sGAG content for an adult mosaic cartilagesample was 199,541±37,371 ug/ml after 12 weeks of explant, while averagesGAG content for a juvenile donor sample was 197,442±15,857 ug/ml after12 weeks of explant, showing that sGAG content has no statisticaldifference between samples from adult and juvenile cartilage tissue.sGAG staining of a mosaic cartilage composition from an adult donorafter 12 weeks of culturing is shown in FIG. 22.

Immunohistochemistry was also performed on mosaic cartilage samplesafter 12 weeks. As shown in FIG. 23, type II collagen was produced bycells that grew out of the mosaic cartilage composition. Type IIcollagen is important for the production of hyaline cartilage, and theformation of the hyaline cartilage outside of the explant shows that themosaic cartilage compositions as described herein can be effective incartilage repair.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, one of skill in the art will appreciate that certainchanges and modifications may be practiced within the scope of theappended claims. In addition, each reference provided herein inincorporated by reference in its entirety to the same extent as if eachreference was individually incorporated by reference.

What is claimed is:
 1. A mosaic cartilage composition comprising: acartilage sheet comprising a plurality of interconnected cartilage tilesthat are separated by channels formed in the cartilage sheet, eachchannel having a respective depth that is less than the maximumthickness of the cartilage sheet; and a biocompatible carrier.
 2. Themosaic cartilage composition of claim 1, wherein the cartilage sheetfurther comprises perforations formed in the cartilage sheet beneath oneor more channels.
 3. The mosaic cartilage composition of claim 1,wherein at least a portion of the cartilage sheet is coated with abiological adhesive.
 4. The mosaic cartilage composition of claim 1,wherein at least a portion of the cartilage sheet is combined withdemineralized bone.
 5. The mosaic cartilage composition of claim 1,wherein at least a portion of the cartilage sheet is combined with abone or cartilage substrate that is seeded with stem cells.
 6. A methodof manufacturing a mosaic cartilage composition, the method comprising:obtaining cartilage tissue from a human cadaveric donor; cutting aplurality of channels into the cartilage tissue, thereby forming acartilage sheet comprising a plurality of interconnected cartilage tilesthat are separated by the channels; and suspending the cartilage sheetin a biocompatible medium.
 7. The method of claim 6, wherein the cuttingstep comprises cutting a plurality of channels into the cartilagetissue, wherein each of the plurality of channels has a depth that isless than the maximum thickness of the cartilage tissue.
 8. The methodof claim 6, further comprising forming perforations in the cartilagesheet beneath one or more channels.
 9. The method of claim 6, whereinthe cutting step comprises cutting the cartilage tissue with a lasercutter, with a mechanical blade, or with a mechanical press.
 10. Themethod of claim 9, wherein the cutting step comprises cutting thecartilage tissue with a laser cutter.
 11. The method of claim 10,wherein the cutting step comprising cutting the cartilage tissue withthe laser cutter at a power from about 0.6 Watt to about 19.5 Watt and afrequency from about 200 Hz to about 2600 Hz.
 12. The method of claim 7,further comprising making perforations in cartilage beneath one or moreof the channels.
 13. The method of claim 6, wherein prior to thesuspending step, the method further comprises coating at least a portionof the cartilage sheet with a biological adhesive.
 14. The method ofclaim 6, wherein prior to the suspending step, the method furthercomprises combining at least a portion of the cartilage sheet withdemineralized bone.
 15. The method of claim 6, wherein prior to thesuspending step, the method further comprises combining at least aportion of the cartilage sheet with a bone or cartilage substrate seededwith stem cells.
 16. A method of treating a cartilage or bone defect ina subject, the method comprising administering to the subject the mosaiccartilage composition of claim
 1. 17. A method of repairing cartilage ina subject, the method comprising administering to the subject the mosaiccartilage composition of claim
 1. 18. A kit comprising the mosaiccartilage composition of claim
 1. 19. The mosaic cartilage compositionof claim 1, wherein the cartilage sheet comprises human cadavericcartilage tissue.
 20. The mosaic cartilage composition of claim 1,wherein the cartilage is obtained from a human adult cadaveric donor age15 years or older.
 21. The mosaic cartilage composition of claim 1,wherein the cartilage is obtained from a human juvenile cadaveric donor.22. The mosaic cartilage composition of claim 1, wherein the cartilageis articular cartilage.
 23. The mosaic cartilage composition of claim 1,wherein the cartilage is non-decellularized cartilage.
 24. The mosaiccartilage composition of claim 1, wherein the cartilage sheet has amaximum thickness of about 0.25 mm to about 5 mm.
 25. The mosaiccartilage composition of claim 1, wherein the thickness of the cartilagesheet beneath the channels is less than about 0.25 mm.
 26. The mosaiccartilage composition of claim 1, wherein at least some of the tiles arecircular shape in shape.
 27. The mosaic cartilage composition of claim26, wherein the tiles that are circular in shape have an averagediameter of about 0.5 mm to about 3 mm.
 28. The mosaic cartilagecomposition of claim 1, wherein at least some of the tiles of are squareor rectangular shape in shape.
 29. The mosaic cartilage composition ofclaim 28, wherein the tiles that are square or rectangular in shape havean average length and/or width of about 0.5 mm to about 3 mm.
 30. Themosaic cartilage composition of claim 1, wherein the tiles aresubstantially uniform in shape and/or size.
 31. The mosaic cartilagecomposition of claim 1, wherein the biocompatible medium comprises acryopreservation medium.
 32. The mosaic cartilage composition of claim1, wherein the cryopreservation medium comprises dimethyl sulfoxide(DMSO) and serum.
 33. The mosaic cartilage composition of claim 3,wherein the biological adhesive is at least one of fibrin, fibrinogen,thrombin, fibrin glue, polysaccharide gel, cyanoacrylate glue,gelatin-resorcin-formalin adhesive, collagen gel, syntheticacrylate-based adhesive, cellulose-based adhesive, basement membranematrix, laminin, elastin, proteoglycans, or autologous glue.
 34. Themethod of claim 6, wherein the human cadaveric donor is an adultcadaveric donor age 15 years or older.
 35. The method of claim 6,wherein the human cadaveric donor is a juvenile donor.
 36. The method ofclaim 6, wherein the cartilage is articular cartilage.
 37. The method ofclaim 6, wherein the cartilage is non-decellularized cartilage.
 38. Themethod of claim 6, wherein the cartilage sheet has a maximum thicknessof about 0.25 mm to about 5 mm.
 39. The method of claim 6, wherein thethickness of the cartilage sheet beneath the channels is less than about0.25 mm.
 40. The method of claim 6, wherein cutting step comprisesforming at least some tiles that are circular in shape.
 41. The methodof claim 40, wherein the tiles that are circular in shape have anaverage diameter of about 0.5 mm to about 3 mm.
 42. The method of claim6, wherein cutting step comprises forming at least some tiles that aresquare or rectangular in shape.
 43. The method of claim 42, wherein thetiles that are a square or rectangular in shape have an average lengthand/or width of about 0.5 mm to about 3 mm.
 44. The method of claim 6,wherein the tiles are substantially uniform in shape and/or size. 45.The method of claim 6, wherein the biocompatible medium comprises acryopreservation medium.
 46. The method of claim 45, wherein thecryopreservation medium comprises dimethyl sulfoxide (DMSO) and serum.47. The method of claim 13, wherein the biological adhesive is at leastone of fibrin, fibrinogen, thrombin, fibrin glue, polysaccharide gel,cyanoacrylate glue, gelatin-resorcin-formalin adhesive, collagen gel,synthetic acrylate-based adhesive, cellulose-based adhesive, basementmembrane matrix, laminin, elastin, proteoglycans, or autologous glue.